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      Use of flow cytometry and total viable count to determine the effects of orange juice composition on the physiology of Escherichia coli

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          Abstract

          Orange juice (OJ) contains numerous compounds some of which are known to play key roles in growth and survival of bacteria. This study aimed to investigate the effects of natural or processing‐induced variations in OJ composition on the physiology of Escherichia coli. OJ and model OJ (MOJ) samples containing various sugars, organic acids, amino acids, or ascorbic acid were inoculated with E. coli K‐12 MG1655 in different growth phases. The culturability, viability, and physiology of the cells were investigated during storage using plate counting and flow cytometry. Generally, stationary‐phase cells displayed the greatest survival in both MOJ and OJ. Increase in incubation temperature from 4 to 22.5ºC caused a significant decrease in both healthy and culturable cell populations. Supplementation of MOJ with ascorbic acid and amino acids increased both the viability and culturability of the cells. Similar trends were observed in amino acid‐supplemented OJ, albeit at a slower rate. In contrast, variations in sugar or organic acid composition had negligible effects on the physiological status of the cells. In summary, natural variation in ascorbic acid or amino acid concentrations could potentially have an adverse effect on the microbiological safety of orange juice.

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          The importance of the viable but non-culturable state in human bacterial pathogens

          Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.
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            Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria

            Under stress conditions, many species of bacteria enter into starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. Several human pathogenic bacteria have been reported to enter into the VBNC state under these conditions. The pathogenic VBNC bacteria cannot be grown using conventional culture media, although they continue to retain their viability and express their virulence. Though there have been debates on the VBNC concept in the past, several molecular studies have shown that not only can the VBNC state be induced under in vitro conditions but also that resuscitation from this state is possible under appropriate conditions. The most notable advance in resuscitating VBNC bacteria is the discovery of resuscitation-promoting factor (Rpf), which is a bacterial cytokines found in both Gram-positive and Gram-negative organisms. VBNC state is a survival strategy adopted by the bacteria, which has important implication in several fields, including environmental monitoring, food technology, and infectious disease management; and hence it is important to investigate the association of bacterial pathogens under VBNC state and the water/foodborne outbreaks. In this review, we describe various aspects of VBNC bacteria, which include their proteomic and genetic profiles under the VBNC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.
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              Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential.

              Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.
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                Author and article information

                Contributors
                t.w.overton@bham.ac.uk
                Journal
                Food Sci Nutr
                Food Sci Nutr
                10.1002/(ISSN)2048-7177
                FSN3
                Food Science & Nutrition
                John Wiley and Sons Inc. (Hoboken )
                2048-7177
                13 August 2018
                October 2018
                : 6
                : 7 ( doiID: 10.1002/fsn3.2018.6.issue-7 )
                : 1817-1825
                Affiliations
                [ 1 ] Bioengineering School of Chemical Engineering The University of Birmingham Birmingham UK
                [ 2 ] Institute of Microbiology & Infection The University of Birmingham Birmingham UK
                [ 3 ]Present address: National Centre for Food Manufacturing Holbeach Technology Park University of Lincoln Holbeach Lincolnshire UK
                Author notes
                [*] [* ] Correspondence

                Tim W. Overton, Bioengineering, School of Chemical Engineering, The University of Birmingham, B15 2TT Birmingham, UK.

                Email: t.w.overton@ 123456bham.ac.uk

                Author information
                http://orcid.org/0000-0003-3050-2549
                Article
                FSN3756
                10.1002/fsn3.756
                6189610
                95a02e49-8e5f-45c0-83b3-463a4a732db4
                © 2018 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 07 June 2018
                : 05 July 2018
                : 10 July 2018
                Page count
                Figures: 4, Tables: 1, Pages: 9, Words: 6169
                Funding
                Funded by: Biotechnology and Biological Sciences Research Council
                Categories
                Original Research
                Original Research
                Custom metadata
                2.0
                fsn3756
                October 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.5.0.1 mode:remove_FC converted:16.10.2018

                amino acids,e. coli,flow cytometry,orange juice,viable but nonculturable

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